Smolke:Protocols/Freezer stocks: Difference between revisions
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**I've never understood the reason for back-diluting before making a freezer stock. You're just adding more time for the plasmid to mutate before being frozen. I make my freezer stocks directly out of my overnight cultures and they work fine. -Josh | **I've never understood the reason for back-diluting before making a freezer stock. You're just adding more time for the plasmid to mutate before being frozen. I make my freezer stocks directly out of my overnight cultures and they work fine. -Josh | ||
***I believe the back-dilution is to ensure that you freeze the cells when they are in the exponential growth phase and are the healthiest. If the stocks made from overnight culture grows just fine I suppose there's no problem with that. --YC | ***I believe the back-dilution is to ensure that you freeze the cells when they are in the exponential growth phase and are the healthiest. If the stocks made from overnight culture grows just fine I suppose there's no problem with that. --YC | ||
***The back-dilutions are to ensure that you make freezer stocks of cells when they are in late exponential phase and healthiest. This way you have more of your cells healthy and alive when you make the freezer stocks (with the idea that they will last longer and grow up better from the freezer stocks). Typically, I always made freezer stocks in the late exponential phase (ODs for this are different for yeast and bacteria). --CDS | |||
*If I had reasons to doubt the sequence accuracy and did not wish to make a lot of freezer stocks before verifying the sequence, I would inoculate from my restreaked plate right after the sequence has been verified (the plate is generally only 2 days old at this point). If I cannot make the freezer stocks before the plate is ~1 week old, I would transform new cells with sequenced plasmids and inoculate from the newly transformed cells. --YC | *If I had reasons to doubt the sequence accuracy and did not wish to make a lot of freezer stocks before verifying the sequence, I would inoculate from my restreaked plate right after the sequence has been verified (the plate is generally only 2 days old at this point). If I cannot make the freezer stocks before the plate is ~1 week old, I would transform new cells with sequenced plasmids and inoculate from the newly transformed cells. --YC | ||
Latest revision as of 20:51, 7 January 2010
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